Polynucleotide phosphorodithioate

ABSTRACT

Oligonucleotides having at least one phosphorodithioate internucleotide linkage. Phosphorodithioate oligonucleotides exhibit stronger antiviral activity than the corresponding phoshorothioate derivatives.

Research leading to the making of the invention described herein wassupported, in part, with federal funds. Accordingly, the United StatesGovernment has certain statutory rights to the invention describedherein.

This application is a continuation of our earlier filed U.S. patentapplication 793,171, filed Nov. 18th 1991, now U.S. Pat. No. 5,278,302;which in turn is a continuation of U.S. Pat. application 545,238 filedJun. 27th 1990, and now abandoned; which in turn is acontinuation-in-part application of our earlier filed U.S. patentapplication 332,247, filed Mar. 31st 1989 and now abandoned; which inturn is a continuation-in-part application U.S. patent application198,886, filed May 26th 1988 and now abandoned.

For the past several years, various nucleoside and nucleotide analogshave been screened for antiviral activity and, in some cases, observedto be effective. This approach has now been extended to the retroviruseswhere it has been found that certain analogs such as3'-azido-2',2'-dideoxythymidine [see Proc. Natl. Acad. Sol. USA 82:7096(1985)] and the 2',2'-dideoxynucleosides [see Proc. Nat. Acad. Sci. USA82:1911 (1986)] are effective antivirals because they inhibit retroviralreplication and reverse transcriptase activity. An alternative approachby other investigators has been to use oligonucleotides or their analogsas antivirals. For this purpose several oligonucleotides andoligonucleotide analogs having methylphosphonate, phosphorothioate, andphosphoroamidate internucleotide linkages have been tested and shown tobe effective antivirals [see Cancer Research 48:2659 (1988)].

Oligonucleotide therapy is being investigated aggressively because, asantivirals, these compounds have known activities in inhibiting primerbinding of reverse transcriptase. They activate reverse transcriptaseRNase H activity; they block translation of viral RNA genes throughhybridization arrest; or they inhibit RNA splicing reactions. Themechanism of inhibition depends upon the choice of oligonucleotideanalog and its nucleotide sequence [see Cancer Research 48:2659 (1988)].

High yielding methodologies are currently available for the rapidsynthesis of sequence defined polynucleotides having the naturalinternucleotide linkage [see Science 230:281 (1995), U.S. Pat. Nos.4,425,732 and 4,458,066], An important step in these methodologies isthe oxidation of the intermediate phosphate triester to the naturallyoccurring phosphate triester with aqueous iodine. These phosphitetriesters can also be oxidized, under anhydrous conditions with aminesor ammonia and iodine, to yield variable reported amounts ofoligonucleotide phosphoramidates, or with sulfur to yieldoligonucleotide phosphorothioates [see Chemica Scripta 26:221 (1986),and Tetrahedron Letters 21:4149 (1980)]. Other methods employingH-I-phosphonate internucleotide linkages can also be used to synthesizeoligonucleotide phosphoramidates and oligonucleotide phosphorothioates[see Tetrahedron Letters 7:5575 ( 1986)]. Oligonucleotidemethylphosphonates are synthesized from nucleosidemethylphosphonamidites [see Tetrahedron 40:95 (1984) and TetrahedronLetters 25:1437 (1984)].

Recently, methods were developed for synthesizing oligonucleotidescontaining phosphorodithioate internucleotide linkages (such as thosedepicted in Examples I, II and III, or in U.S. Pat. No. 5,218,103, thedisclosure of which is incorporated in toto herein). These developmentshave now led to the discovery of the present invention thatphosphorodithioate-containing oligonucleotides, a new class of antiviralchemotherapeutic agents, are inhibitors of viral reverse transcriptases.

In general, the oligonucleotide phosphorodithioates according to thepresent invention, can be represented by the formulae I and II: ##STR1##wherein R is H or a blocking group; A is H, OH, halogen, SH, NH₂, orazide; B is a nucleoside or deoxynucleoside base (including purines,e.g. adenine, hypoxanthine, guanine, or their derivatives, andpyrimidines, e.g., cytosine, uracil, thymine, or their derivatives)which may be the same or different at each occurrence in the compound; nis an integer from zero to thirty; and m is an integer from one tothirty. If the repeat units represented by m and n are within the sameoligonucleotide phosphorodithioate, it is understood that tile repeatunits contained within m and n can be positioned in any sequence andthat the sum of m and n usually would not exceed thirty. Morespecifically, these formulae are intended to include any permutation ofphosphorodithioate and normal diester linkages. Thus, these formulaeshould be interpreted as encompassing a series of dithioate linkages (n)followed by a series of normal phosphate diester linkages (m) orencompassing a series of alternating or interspersed phosphorodithioatelinkages within art oligonucleotide polymer. Accordingly, for clarity ofdisclosure the compounds of the present invention may also begenerically depicted as an oligonucleotide having at least onephosphorodithioate linkage substituted for the normally occurringphosphate diester linkage in the oligonucleotide. That is,oligonucleotides according to the present invention can be representedby the formula:

    R-O-[N-O-L-O-N].sub.t -O- R

wherein N represents a nucleoside moiety (that is a purine or pyrimidinebase in glycosidic linkages with a sugar) of the formula ##STR2##wherein A, B and R are as defined previously; wherein L is a phosphateinternucleotide linkage of the formula ##STR3## wherein at least one Lin the formula is ##STR4## and wherein t is an integer from 1 to 60,preferably from 1 to 30.

The new class of chemotherapeutic compounds according to the presentinvention and represented by the formulae above are oligonucleotideshaving a 3'-5' phosphate diester linkage, ribose and deoxyribose sugars,purine and pyrimidine bases, and the nucleosides and deoxynucleosideslinked to phosphorus through oxygen covalently joined at the 3' and5'-carbons of the sugars. Compound I has two sulfur atoms bonded to eachphosphorus whereas compound II has at least one phosphorus bonded to twosulfur atoms and one phosphorus bonded to two oxygens while eachremaining phosphorus is bonded to either two sulfurs or two oxygens.Thus it can be seen that compound I depicts an oligonucleotide havingexclusively phosphorodithioate internucleotide linkages whereas compoundII depicts an oligonucleotide having at least one phosphorodithioateinternucleotide linkage and one phosphate internucleotide linkage withthe remainder being either phosphorodithioate or naturally occurringphosphate diester linkages. In each of Formula I or II, B may be thesame or different base for each occurrence.

The chemical synthesis of compound I is completed using appropriatelyprotected deoxynucleoside or nucleoside phosphorothioamidites assynthons, preferably a deoxynucleoside or nucleoside joined covalentlyto a silica support. Activation of the synthon is most easilyaccomplished with tetrazole. The reaction sequence is then completed byoxidation with sulfur, acylation of unreacted, silica bondeddeoxynucleoside or nucleoside, and the subsequent selective removal ofappropriate protecting groups. This cycle can then be used repetitivelyin order to extend the oligonucleotide so that it contains as many as 32nucleosides (i.e., when n equals 30).

A similar sequence may be used to prepare compound II. In this sequence,two synthons, a deoxynucleoside or nucleoside phosphoramidite and adeoxynucleoside or nucleoside phosphorothioamidite, are used to preparean oligonucleotide having the phosphate and phosphorothioateinternucleotide linkages with m plus n equal to thirty.

In order to provide a more detailed understanding of the presentinvention, the following examples and procedures are provided. Thesedepict the formation of compounds I and II, demonstrate how thesecompounds inhibit viral reverse transcriptases, and provide a morecomplete understanding and illustration of the present invention. Theyare, however, examples, and as such are not intended in any manner tolimit the scope of the present invention.

The procedure outlined in the following Example I may also be used toproduce dipyrrolidinylchlorophosphine, and bis(dimethylamino)chlorophosphine. Preparation of thiophosphoramidites of the formula##STR5## wherein B may be 1-thyminyl, 1-(N-4-benzoylcytosinyl),9-(N-6-benzoyladeninyl), or 9-(N-2-isobutyrylguaninyl); DMT isdi-p-anisylphenylmethyl (dimethoxytrityl); M may be S-(4-chlorobenzyl)or S-(2,4-dichlorobenzyl); and X may b N,N-dimethylamino or pyrrolidinylwherein the further use of these compounds to prepare oligonuoleotideshaving phosphorodithioate internucleotide linkages are presented in theremaining examples.

EXAMPLE I

Bis(dimethylamino)chlorophosphine was prepared by addingtris(dimethylamino)-phosphine (36.3 ml, 32.6 g, 0.2 mole) andtrichlorophosphine (8.7 ml, 13.7 g, 0.1 mole) to anhydrous ether (100ml). After stirring for 3 hours at room temperature, solvent was removedby concentration in vacuo at room temperature. The product was thendistilled (b.p. 72°-75 ° C.) at reduced pressure (approx. 16 mm Hg)using a water aspirator to yield 30 g. of product.

Example II describes the synthesis of5'-O-dimethoxytrityl-N4-benzoyldeoxycytidyl, 1-3'-S(4-chlorobenzyl)phosphorothiopyrrolidinite, and its further use to prepareoligonucleotides having phosphorodithioate internucleotide linkages. Thesame procedure can be used for the other suitably protecteddeoxynucleosides. Similarly the same procedure is useful for all the2,4-dichlorobenzyl and 4-chlorobenzyl protected sulfur derivatives ofthe N,N-dimethylamino and pyrrolidinyl amidites. Table I summarizes the³¹ P-NMR data for all these amidites.

Using the deoxycytidine phosphorothioamidite made in the followingExample II, Compound Ia, wherein n is 12, B is cytosine and A ishydrogen was prepared. Compound Ia therefore has the following structurewhere C represents deoxycytidine and x represents the phosphorodithioateinternucleotide linkage.

    d(CxCxCxCxCxCxCxCxCxCxCxCxCxC)

EXAMPLE II

5'-O-Dimethoxytrityl-N4-benzoyldeoxycytidine (317 mg, 0.5 mmol) wasdissolved in a mixture of acetonitrile (2 ml) and triethylamine (1 ml)under argon. Bispyrrolidinylchlorophosphine (124 mg, 0.6 mmol) was addedwhich was followed by the immediate formation of a precipitate. After 5minutes stirring at room temperature, 4-chlorobenzylmercaptan (159 mg, 1mmol) was added to the reaction mixture and the solution, including theprecipitate, was concentrated to a glass in vacuo at room temperature.The glass was resuspended in acetonitrile (2 ml). The ³¹ P-NMR spectrumof the reaction mixture indicated that the major phosphorus containingproduct was the diastereoisomers of the thioamidite (161.5, 159.7 ppm).Minor impurities were an adduct of bispyrrolidinylchlorophosphine and4-chlorobenzylmercaptan (107.0 ppm) and hydrolysis products (12.4 ppm).Triethylamine was next added to the reaction mixture. The solution wasdiluted with deacidified ethylacetate (50 ml) and extracted with aqueoussaturated sodium bicarbonate (50 ml×2) and brine. The combined aqueoussolutions were back-extracted with deacidified ethylacetate (10 ml). Theorganic solutions were combined, dried for 1 hour over sodium sulfate inthe presence of 10% (volume) triethylamine, filtered, and the filtercakewashed with 5 ml deacidified ethylacetate. The organic solution was thenconcentrated in vacuo to a white foam. This foam was dissolved intoluene (10 ml) containing 1% triethylamine and the product isolated byprecipitation into n-pentane:triethylamine (999:1, v/v). Afterfiltration, the product was dried in vacuo over phosphorus pentoxide andpotassium hydroxide and isolated in 83.1% yield (741 mg).

Using a deoxynucleoside attached covalently to a silica based polymersupport through the 3'-hydroxyl (in accordance with the teaching of U.S.Pat. No. 4,458,066, the disclosure of which is incorporated herein),synthesis of deoxyoligonucleotides containing phosphorodithioatelinkages proceeded according to the reaction sequence outlined belowwherein (P) represents the polymer support. ##STR6## Wherein R₁ is ablocking group. More specifically, the over-all reaction sequence forthe making of the present invention is depicted as: ##STR7## wherein Ris a protecting group as shown in the following examples, B is anucleoside or deoxynucleotide base, and (P) is a silica-based support asdefined below.

In general, synthesis began by reacting a dry acetonitrile solution ofany thiophosphoramidite according to Example II (10 equivalents) andtetrazole (50 equivalents) with 1 μmole of deoxynucleoside on silica for30 seconds (i) followed by a 400 sec oxidation with 5% sulfur inpyridine:carbon disulfide (1:1, v/v) (ii). Coupling was performed twiceto ensure high yields (greater than 98%). Acylation of unreacteddeoxynucleoside (iii), detritylation (iv) and various washes were thesame as those described previously for synthesizing natural DNA fromdeoxynucleoside phosphoramidites (U.S. Pat. No. 4,415,732 and Science230:281 (1085)). Repetitions of this cycle an additional twelve timesled to the synthesis of compound Ia. Deoxyoligonucleotides such ascompound II having both phosphorodithioate and phosphate internucleotidebonds may be synthesized when both deoxynucleoside phosphorothioamiditesand deoxynucleoside phosphoramitides are used during synthesis.

Synthetic deoxyoligonucleotides were isolated free of protecting groupsusing a two-step protocol (thiophenol:t-ethylamine:dioxane, 1:1:2, v/v/vfor 24 h followed by conc. ammonium hydroxide for 15 h), and thenpurified to homogeneity by standard procedures (polyacrylamide gelelectrophoresis and reverse phase hplc). 31 P-NMR spectra ofphosphorodithioate DNA indicated that this synthesis protocol yieldedDNA containing phosphorodithioate internucleotide linkages.

                  TABLE I                                                         ______________________________________                                        31P-NMR Characterization of                                                   Deoxynucleoside Phosphorothioamidites                                         Base    Amine        Mercaptan     .sup.31 P-NMR.sup.a                        (B)     (X)          (M)           (δ)                                  ______________________________________                                        T       pyrrolidinyl 2,4-dichlorobenzyl                                                                          164.8;161.8                                T       pyrrolidinyl 4-chlorobenzyl                                                                              164.2;161.0                                T       dimethylamino                                                                              4-chlorobenzyl                                                                              172.3;170.5                                T       dimethylamino                                                                              2,4-dichlorobenzyl                                                                          172.1;170.4                                C.sup.Bz                                                                              pyrrolidinyl 2,4-dichlorobenzyl                                                                          165.1;162.6                                C.sup.Bz                                                                              pyrrolidinyl 4-chlorobenzyl                                                                              161.8;159.9                                C.sup.Bz                                                                              dimethylamino                                                                              4-chlorobenzyl                                                                               1.71;170.7                                C.sup.Bz                                                                              dimethylamino                                                                              2,4-dichlorobenzyl                                                                          172.0;171.0                                A.sup.Bz                                                                              pyrrolidinyl 2,4-dichlorobenzyl                                                                          163.8;162.7                                A.sup.Bz                                                                              pyrrolidinyl 4-chlorobenzyl                                                                              163.5;162.3                                A.sup.Bz                                                                              dimethylamino                                                                              4-chlorobenzyl                                                                              171.8;170.9                                A.sup.Bz                                                                              dimethylamino                                                                              2,4-dichlorobenzyl                                                                          171.7;170.9                                G.sup.Bz                                                                              pyrrolidinyl 2,4-dichlorobenzyl                                                                          163.9;160.9                                G.sup.Bz                                                                              pyrrolidinyl 4-chlorobenzyl                                                                              163.4;161.6                                G.sup.Bz                                                                              dimethylamino                                                                              4-chlorobenzyl                                                                              171.5;169.5                                G.sup.Bz                                                                              dimethylamino                                                                              2,4-dichlorobenzyl                                                                          171.9;169.6                                ______________________________________                                         .sup.a indicates .sup.31 PNMR were recorded on a Brucker WM250 with 85%       aqueous H.sub.3 PO.sub.4 as external standard.                                T, C.sup.Bz, A.sup.Bz, and G.sup.iB refer to thymine, Nbenzoylcytosine,       Nbenzoyladenine, and Nisobutyrylguanine respectively;                         R1 is dimethyltrityl,                                                         A is hydrogen                                                            

Syntheses are described in the following Example III for Compounds IIa,IIb and IIc wherein m and n are variables for IIa, IIb, and IIc, R ishydrogen, B is cytosine, and A is hydrogen. Compounds IIa, IIb and IIchave the following structures wherein C represents deoxycytidine, xrepresents the phosphorodithioate internucleotide linkage, and p tilenaturally occurring phosphate internucleotide linkage.

    IIa: d(CpCxCpCpCpCpCpCpCpCpCpCpCxCpC)

    IIb: d(CpCpCpCpCpCpCxCpCpCpCpCpCpCpC)

    IIc: d(CxCpCxCpCxCpCxCpCxCpCxCpCxCpC)

Synthesis of Dinucleoside Phosphorodithioate Triesters of the formula:##STR8## wherein B may be 1-thyminyl, 1-(N-4-benzoylcytosinyl),9-(N-6-benzoyladeninyl), or 9-(N-2-isobutyrylguaninyl); DMT isdi-p-anisylphenylmethyl (dimethoxytrityl); and Ac is acetyl and thefurther conversions of the deoxydicytidine derivative to deoxycytidineoligodeoxynucleotides having phosphorodithioate internucleotide linkagesat various positions are presented in this example.

EXAMPLE III

A. The synthesis of a thymidine dinucleotide having a phosphorodithioateInternucleotide linkage is described below:

5'-O-dimethoxytritylthymidine (1.2 g. 2.21 mmol) was dried byco-evaporation with anhydrous THF and then dissolved in THF (10 ml) andtriethylamine (0.46 ml, 3.3 mmol).Bis-(diisopropylamino)-chlorophosphine (650 mg, 2.44 mmol) was added andthe solution stirred at room temperature. After 35 minutes, theprecipitate was removed by filtration and washed with THF (1 ml). Thecombined filtrates containing the deoxynucleoside phosphorodiamiditewere pooled, concentrated in vacuo, and redissolved in acetonitrile (5ml). 3'-O-acetylthymidine (639 mg, 2.25 mmol) and tetrazole (142 mg, 2.0mmol) were dried by co-evaporation with THF (10 ml), redissolved inacetonitrile (5 ml), and added to the acetonitrile solution of thedeoxynucleoside phosphorodiamidite. After stirring for 45 minutes atroom temperature, the reaction mixture was diluted with dichloromethane(75 ml), extracted with an aqueous sodium bicarbonate solution (5% w/v),dried over sodium sulfate, filtered, and concentrated in vacuo to a gum.The product was then purified by column chromatography (100 ml silica,ethylacetate :dichloromethane :-triethylamine; v/v/v) to yield 1.59 g ofthe deoxydinucleoside phosphoramidite (1.66 mmol, 75%).

The deoxynucleoside phosphoramidite was then converted to thedeoxydinucleoside phosphorodithioate triester. The deoxydinucleosidephosphoramidite (1.59 g, 1.66 mmol) was dissolved in acetonitrile (7ml). 4-chlorobenzylmercaptan (1.0 ml, 1.2o g, 7,6 retool) and tetrazole(281 mg, 4.01 mmol) were added and the reaction mixture stirred at roomtemperature for 30 minutes. A solution of sulfur in toluene:2,6-lutidine(19:1, v/v, 10 ml containing 4 mmol atomic sulfur) was added and theresulting solution stirred for 10 minutes. The reaction mixture wasdiluted with ethylacetate (75 ml), extracted with an aqueous sodiumbicarbonate solution (5%, w/v), dried over sodium sulfate, filtered andconcentrated in vacuo to an oil. The oil was dissolved in ethylacetate(40 ml) and triturated with hexanes (200 ml) to give a crude product asa white powder. Purification by silica column chromatography (100 mlsilica, 2-12% methanol in dichloromethane as % eluant) yields thedeoxydinucleoside phosphorodithioate triester (1.59 g, 1.52 retool,91%).

Removal of the 3'-O-acetyl group (0.15M t-butylamine in methanol, 0 °C., 10 h) yields a deoxydinucleoside phosphorodithioate that can be usedfor DNA synthesis (1.26 g, 1.28 mmol, 84%). The deoxydinucleosidephosphorodithioate is converted to the 3'-phosphoramidite and then usedto synthesize DNA on a polymer support.

B. The synthesis of deoxycytidine oligomers containingphosphorodithioate internucleotide linkages is described below:

5'-O-DimethoxytrityI-N-toluoyldeoxycytidine was prepared by a minormodification of a published procedure (Tetrahedron 37:363 (1981)).Deoxycytidine hydrochloride (10 mmol, 2.64 g) was co-evaporated twicewith anhydrous pyridine and resuspended in pyridine (50 ml).Trimethylchlorosilane (7.5 ml, 59 mmol) was added and the mixturestirred for 45 minutes at room temperature. o-Toluoyl chloride (1.44 ml,11 mmol) was added and the reaction stirred for two additional hours.The reaction mixture was chilled in an ice bath, treated with methanol(10 ml) and 25% ammonium hydroxide (20 ml) for 30 min, and thesuspension removed by filtration. The resulting solution wasconcentrated to dryness in vacuo. The resulting solid was suspended in40 ml dichloromethane:methanol (8:2) and the insoluble salts removed byfiltration. The filtrate was concentrated in vacuo to an oil,reconcentrated twice in vacuo after addition of pyridine and redissolvedin pyridine (50 ml). After addition of 0.9 equivalents ofdimethoxytrityl chloride (3.05 g), the reaction mixture was stirred for30 rain at 0 ° C. and 30 rain at room temperature.Dimethoxytritylchloride (0.3 equivalents) was added and stirring wascontinued for 30 minutes. The reaction was quenched by addition ofmethanol (1 ml) and the solution concentrated in vacuo. The resultingoil was dissolved in dichloromethane (75 ml) and extracted sequentiallywith aqueous 5% sodium bicarbonate (w/v) and brine. The combined organicphase was dried over sodium sulfate, filtered, concentrated to drynessin vacuo, dissolved in dichloromethane:pyridine (99.5:0.5, v/v) and theproduce purified by column chromatography (50 g silica,dichloromethane:methanol:pyridine gradient from 0 to 3% methanol; 400 mleach). Fractions containing the5'-O-dimethoxytrityl-N-toluoyldeoxycytidine were pooled, concentrated invacuo, redissolved in ethylacetate and precipitated into pentane (5.01g, 7.7 mmol, 77%).

3'-O-Phenoxyacetyl-N-toluoyldeoxycytidine was prepared by minormodification of a published procedure (Tetrahedron Letters 4273 (1968)).5'-O-DimethoxytrityI-N-toluoyldeoxycytidine (1.94 g, 3 mmol) andphenoxyacetic anhydride (1.72 g, 6 mmol) was dissolved intetrahydrofuran (50 ml). After addition of pyridine (173 ml, 9 mmol),the solution was stirred for 14 hours at room temperature and thenconcentrated in vacuo. The resulting oil was dissolved indichloromethane (75 ml), extracted twice with 5% aqueous sodiumbicarbonate (100 ml, w/v) and the combined aqueous phases extracted withdichloromethane (50 ml). The product in the combined organic phase wasdried over sodium sulfate, filtered, concentrated to dryness in vacuo,redissolved in ethylacetate and precipitated in pentane. The solidcorresponding to totally protected deoxycytidine was dissolved indichloromethane: methanol (8:2, v/v) and chilled in an ice bath. Asolution of p-toluenesulfonic acid (2121 g, 12 mmol) indichloromethane:methanol (50 ml, 8:2, v/v) was added and the solutionstirred for one hour in an ice bath. The reaction was then quenched byaddition of 5% aqueous sodium bicarbonate. The organic layer wasextracted with brine and the aqueous phase re-extracted withdichloromethane (60 ml). The combined organic phase was dried oversodium sulfate, filtered and concentrated to dryness in vacuo. Theresulting oil was dissolved in dichloromethane and the product purifiedby silica gel column chromatography (20 g of silica, elution withdichloromethane and dichloromethane:methanol (1 to 3% methanol).Fractions containing 3'-O-phenoxyacetyl-N-toluoyldeoxycytidine werepooled, concentrated to an oil, and the product isolated as aprecipitate by addition of ethylacetate (1.20 g, 83%).

Deoxydicytidine phosphoramidite in protected form was prepared using thefollowing procedure:

5'-O-DimethoxytrityI-N-toluoyldeoxycytidine (647 mg, 1 mmol) wasco-evaporated three times with THF, dissolved in THF (5 ml) andtriethylamine (0.21 ml, 1.5 mmol) and reacted with bis(N,N-diisopropylamino) chlorophosphine (320 mg, 1.2 mmol). After 90minutes under argon, the reaction mixture was filtered under argonpressure to remove insoluble salts. The salts were washed with THF (2ml). The filtrate was concentrated to dryness and the productredissolved in acetonitrile (2 ml).3'-O-phenoxyacetyl-N-toluoyldeoxycytidine (527 mg, 1.1 mmol) andtetrazole (70 mg, 1 mmol) were suspended in acetonitrile (4 ml) and theabove solution, including 1.5 ml acetonitrile used to wash the flask,was added. The reaction mixture was stirred under argon for 105 min. andthen poured into ethylacetate:triethylamine (99:1, v/v, 25 ml), theorganic phase was dried over sodium sulfate, filtered, and concentratedin vacuo. Purification was achieved by silica gel column chromatography(25 g silica, elution with hexanes:dichloromethane:triethylamine,50:50:0.5, 400 ml, 45:55:0.5, 200 ml, 40:60:0.5, 200 ml, and 35:65:0.5,100 ml). Product fractions were pooled, concentrated in vacuo, andprecipitated into pentane (67%).

Deoxydicytidine phosphorodithioate was prepared using the followingprocedure:

The deoxydicytidine phosphoroamidite as prepared in the previousprocedure (1.4.0 g, 1.12 retool) was dissolved in acetonitrile (5 ml)(previously flushed with helium to avoid oxygen oxidation ofthiophosphite) and 4-chlorobenzyl-mercaptan (0.5 ml, 3.7 mmol) andtetrazole (190 mg, 2.7 retool) were added. The solution was stirredunder argon for 30 min and, without isolation, the resultingthiophosphite was oxidized to tile phosphorodithioate triester byaddition of 5 ml of a 0.4M solution of sulfur in toluene:lutidine (19:1,v/v). Based on 31 P-NMR analysis, oxidation was complete after 10minutes. The reaction mixture was diluted with ethylacetate (75 ml),extracted twice with 5% aqueous sodium bicarbonate (75 ml each), and thecombined aqueous phases back-extracted with ethylacetate (50 ml). Thecombined organic phases were dried over sodium sulfate, filtered, andconcentrated in vacuo to an oil. The oil was dissolved in a minimalamount of dichloromethane, diluted with ethylacetate to approximately 40ml, and the product precipitated by addition of 200 ml hexanes. Thewhite precipitate was filtered, redissolved in dichloromethane, and thesolution concentrated to dryness. The product was purified by silica gelcolumn chromatography (40 g silica gel, elution withdichloromethane:hexanes:triethylamine, 66:33:0.03, 400 ml anddichloromethane:triethylamine, 100:0.03, 200 ml). Fractions containingthe completely protected product were pooled, concentrated in vacuo,redissolved in dichloromethane, and precipitated into pentane (60%).

The 3'-O-phenoxyacetyl protecting group was removed using the followingprocedure:

The completely protected deoxydicytidine phosphorodithioate triester(355 mg, 0.264 mmol) was dissolved in acetonitrile (3 ml) and dilutedwith methanol (9 ml). After chilling the solution in an ice bath,t-butylamine in methanol(0.3M, 12 ml) was added and the reaction mixturestirred for 90 rain in an ice bath. The reaction solution wasconcentrated to dryness and the product purified by silica gel columnchromatography (30 g silica, elution with dichloromethane:triethylamine,100:0.03, 100 ml followed by 200 ml each ofdichloromethane:methanol:triethylamine, 99:1:0/03, 98:2:0/03 and97:3:0.03). Product fractions were concentrated to dryness, redissolvedin dichloromethane, and precipitated into pentane (95% yield).

The deoxydicytidine phosphorodithioate was next converted to the3'-phosphoramidite which is useful as a synthon for synthesizing DNAcontaining dithioate internucleotide linkages. The deoxydicytidinephosphorodithioate having a free 3'-hydroxyl (304 mg, 0.251 mmol) wasdissolved in acetonitrile (5 ml).Bis(diisopropylamino)-β-cyanoethoxyphosphine (1 21 mg, 0.402 mmol) andtetrazole (20 mg, 0.286 mmol) were added under argon and the solutionstirred for 2 hours. After quenching with ethylacetate:triethylamine(19.5:0.5) and diluting further with ethylacetate (20 ml), the reactionmixture was extracted twice with 2M triethylammonium bicarbonate (13 mleach) and the aqueous phase back-extracted withethylacetate:triethylamine (19.5:0.5). The organic layer was dried oversodium sulfate, filtered, and concentrated to an oil in vacuo. Theresulting oil was redissolved in dry ethylacetate and precipitated intopentane (87% yield).

Deoxycytidine pentadecamers containing phosphorodithioateinternucleotide linkages at selected sites were synthesized using thedeoxydicytidine phosphorodithioate synthons having a3'-O-(β-cyanoethyl)-N,N-diisopropylphosphoramidite moiety as describedabove and5'-O-dimethoxytrityl-N-benzoyldeoxycytidine-3'-O-(β-cyanoethyl)-N,N-diisopropylphosphoramidite.The standard phosphoramidite synthesis methodology was used (U.S. Pat.Nos. 4,415,732 and 4,458,066). The average coupling efficiency was setat 99% (3 minute coupling time, 0.2 μmol deoxycytidine on controlledpore glass as a support). The products were freed of protecting groupsby treatment with a solution of thiophenol:triethylamine:dioxane (1:1:2,v/v/v) at room temperature for 6 hours (some product remains as theS-protected dithioate (5-10%) when analyzed by gel electrophoresis andconcentrated ammonium hydroxide at 55 ° C. (15 hours). Purification ofthe final product was by either polyacrylamide gel electrophoresis orhigh performance liquid chromatography. Using deoxycytidine as oneembodiment of the present invention, three pentadecamers havingphosphorodithioate linkages at specific positions were synthesized andhave the following sequence:

    d(CpCxCpCpCpCpCpCpCpCpCpCpCxCpC)

    d(CpCpCpCpCpCpCxCpCpCpCpCpCpCpC)

    d(CxCpCxCpCxCpCxCpCxCpCxCpCxCpC)

wherein x represents a dithioate linkage and p represents the naturallyoccurring phosphate internucleotide linkage.

The ability of deoxyoligonucleotide homopolymers made in accordance withthe present invention to inhibit viral reverse transcriptases was testedusing an assay whereby a deoxyoligonucleotide primer (P) was extendedenzymatically using a reverse transcriptase enzyme, deoxynucleotidetriphosphates (dNTP), and a deoxyoligonucleotide as template (T). Thesystem is as follows:

    __________________________________________________________________________      P:   5'-GpApTpTpCpApGpCpTpApGpTpCpCpA                                         T:   3'-CpTpApApGpTpCpGpApTpCpApGpGpTpApGpCpApTpApGp                        TpGpTpCpApApApC                                                                                dATP, dTTP, dCTP, dGTP                                                        Reverse transcriptase                                                         ± deoxyoligonucleotide inhibitor                          P: 5'-GpApTpTpCpApGpCpTpApGpTpCpCpApTpCpGpTpApTpCpApCpAp                      GpTpTpTpG                                                                     T: 3'-CpTpApApGpTpCpTpApTpCpApGpGpTpApGpCpApTpApGpTpGpTp                      CpApApApC                                                                     __________________________________________________________________________

Thus it can be seen that the assay involves DNA repair synthesis.Deoxynucleotide triphosphates are incorporated into the primer strandusing reverse transcriptase as the DNA polymerizing enzyme. Two reversetranscriptases, the human immunodeficiency virus type I reversetranscriptase (HIV-I reverse transcriptase) and avian myeloblastosisvirus reverse transcriptase (AMV reverse transcriptase), and a normalcellular polymerase, the large fragment of E. coli DNA polymerase I(Klenow polymerase), were used in this assay. Severaldeoxyoligonucleotide homopolymers having combinations ofphosphorodithioate internucleotide linkages (Ia, IIa, IIb, IIc, V andVI), one deoxyoligonucleotide homopolymer having phosphorothioateinternucleotide linkages (IIIa) , and one deoxyoligonucleotidehomopolymer having natural phosphate diester linkages (IVa) were testedas inhibitors of the reverse transcriptases. Additionaldeoxyoligonucleotides with heterosequences and having phosphorodithioate(VII, VIII and IX) or phosphorothioate (X) internucleotide linkages werealso tested as inhibitors of the reverse transcriptases. Compounds Ilia,X, and IVa were prepared using published procedures (U.S. Pat. Nos.4,415,731 and 4,458,066; JACS 106:6077 (1984); and Biochemistry 3443(1984)). These compounds have the following sequences whereinternucleotide linkages are represented by x for phosphorodithioate, pfor the naturally occurring phosphate, and for the phosphorothioate:

    Ia: d(CxCxCxCxCxCxCxCxCxCxCxCxCxC)

    IIa: d(CpCxCpCpCpCpCpCpCpCpCpCpCxCpC)

    IIb: d(CpCpCpCpCpCpCpCpCpCpCpCpCpCpC)

    IIc: d(CxCpCxCpCxCpCxCpCxCpCxCpCxCpC)

    IIIa: d(C-C-C-C-C-C-C-C-C-C-C-C-C-C-C)

    IVa: d(CpCpCpCpCpCpCpCpCpCpCpCpCpC)

    V: d(TxTxTxTxTxTxTxTxTxTxTxTxTxT)

    VI: d(AxAxAxAxAxAxAxAxAxAxAxAxAxA)

    VII: d(GxAxTxTxCxAxGxCxTxAxGxTxCxCxA)

    VIII: d(GxCxTxAxCxGxGxCxTxCxGxCxTxG)

    IX: d(CxTxGxTxTxCxGxGxGxCxGxCxCxA)

    X: d(C-T-G-T-T-C-G-G-G-C-G-C-C-A)

The primer (P) and template (T) deoxyoligonucleotides were alsosynthesized using published procedures (U.S. Pat. Nos. 4,415,732 and4,458, 066).

Assays for measuring the inhibition of DNA repair synthesis withphosphorodithioate containing DNA were completed using the followingprocedure:

EXAMPLE IV

Primer (12 μM) and template 10 μM) in a solution of Tris hydrochloride(Tris. HCl, 50 mM, pH 8.3), MgCl₂ (10 mM), and dithiothreitol (DTT, 5mM) were warmed at 90 ° C. for five minutes and then cooled on ice to 0° C. 5'-³² P labeled primer was approximately 0.5% of total primer.Aliquots of primer-template were then mixed with other components toyield assay solutions (20 μl) having the following composition: template(1 μM), primer (1.2 μM), tris-HCl (50 mM, pH 8.3), MgCl₂ (10 mM), KCI(50 mM), DTT (5 mM), dTTP (250 μM), dCTP (250 μM), dGTP (250 μM), andinhibitor oligonucleotide at variable concentrations from zero to 70 μM.Reactions were started by adding AMV reverse transcriptase (7.2 nM),HIV-I reverse transcriptase (10 nM or 50 nM) or Klenow fragment (200nM). Assays were incubated at 37 ° C. for 15 minutes, quenched by addingformamide to 50%, and analyzed by electrophoresis on a 15% denaturingpolyacrylamide gel. Radioactive bands containing polymerized primer andunextended primer were cut from the gels, dried and analyzed in ascintillation counter. The results are presented in Table 2.

                  TABLE 2                                                         ______________________________________                                        SUMMARY OF THE ID.sub.50 VALUES FOR                                           PHOSPHOROTHIOATED DEOXYOLIGONUCLEOTIDES                                                          ID.sub.50 Values                                                  HIV-I Reverse                                                                             AMV Reverse Klenow                                         Inhibitor                                                                            Transcriptase                                                                             Transcriptase                                                                             Fragment                                       ______________________________________                                        Ia     60      nM      250   nM    >>800   nM*                                IIa    30      μM         ND            ND                                 IIb    75      μM         ND            ND                                 IIc    2       μM   11    μM         ND                                 IIIa   2       μM   42    μM         ND                                 IVa    >36     μM** >70   μM*        ND                                 V      30      nM                                                             VI     75      nM                                                             VII    10      nM                                                             VIII   10      nM                                                             IX     4.4     nM                                                             X      126     nM                                                             ______________________________________                                         ND indicates the result was not determined;                                   ID.sub.50 indicates the concentration of inhibitor where the reaction         proceeds to 50% of the uninhibited reaction;                                  *indicates that inhibition was observed at these concentrations whereas       HIVI reverse transcriptase was completely inhibited; and                      **indicates that only 7% inhibition at 36 μM of inhibitor IVa         

The results listed in Table 2 can be summarized as follows. Compound la,a phosphorodithioate containing deoxyoligocytidine, is a very potentinhibitor of HIV-I reverse transcriptase (ID₅₀ =60 nM) and is about 33fold more inhibitory than Ilia, a phosphorothioate linkeddeoxyoligocytidine of about the same length. Similarly 1a inhibits asecond reverse transcriptase, AMV reverse transcriptase, approximately168 fold more effectively than IIIa. Compounds V and VI which correspondto the dithioate derivatives of oligodeoxythmidine (V) andoligodeoxyadenosine (VI) are also very potent inhibitors of HIV reversetranscriptase. The oligodeoxythymidine derivative (V) is even a morepotent inhibitor than the corresponding oligodeoxycytidine derivative(Ia). Also of considerable interest was the discovery that la did notinhibit a normal cellular polymerase, the large fragment of E. coli DNApolymerase I or Klenow fragment, even at 800 nM. At this concentration,HIV-I reverse transcriptase is completely inhibited. Two otherdiscoveries merit comment. First normal deoxyoligocytidine, compoundIVa, is noninhibitory at concentrations where both HIV-I reversetranscriptase and AMV reverse transcriptase are completely inhibited byIa. Also a comparison of the results with HIV-I reverse transcriptaseand Ia, IIa, IIb, and IIc shows that the extent of inhibition correlatesdirectly with the number of phosphorodithioate linkages present in thedeoxyoligonucleotide.

Each of the compounds designated V to IX are either 14 or 15 nucleotidesin length, and all have exclusively dithioate internucleotide linkages.Compounds V and VI are homopolymers having polydeoxythymidine andpolydeoxyadenosine sequences, respectively. Compound IX is of specialsignificance as it has a sequence identical to tile corresponding humanlysine transfer RNA that is used naturally by the HIV reversetranscriptase to initiate viral RNA synthesis [see Cell 40:9 (1985)].The ID₅₀ value for compound IX (4.4 nM) represents essentially 50%inhibition of the total HIV reverse transcriptase in the reactionmixture. Thus, this indicates that the enzyme is being titrated in thetest system and therefore a much lower concentration of compound IX canbe used with a continued inhibitory effect. Compound X has the samesequence as IX but contains all phosphorothioate internucleotidelinkages. As can be seen from the data in Table 2, compound IXcontaining all dithioate linkages is at least 30 fold more inhibitorythan X which has the thioate linkages. Sequences VII and VIII correspondto the primer sequence as used in this assay (VII) and anoligonucleotide (VIII) having tire same base composition as IX but adifferent sequence. As can be seen from the data in Table 2, both VIIand VIII are less inhibitory than IX. Thus this data shows that the DNAsequence corresponding to human lysine transfer RNA (IX), the RNA thatbinds to the primer binding site on the HIV genome, and is used toinitiate DNA synthesis, is the most inhibitory dithioate containingdeoxyoligonucleotide.

These results demonstrate that we have discovered a new class of potentchemotherapeutic agents for the treatment of viruses. These reagents arethe phosphorodithioate containing deoxyoligonucleotides which arestrongly inhibitory toward reverse transcriptases with ID50 values lessthan the 60 nM range. This means that these reagents are at least 33fold more inhibitory than the phosphorothioate class ofoligonucleotides. As was the case with phosphorothioate oligonucleotideswhich are also inhibitory against HIV-I reverse transcriptase [seeCancer Research 48:2659 (1988), and Biochemistry 28:1340 (1989)], it isto be expected this new class of chemotherapeutic agents, thephosphorodithioate oligonucleotides, to be inhibitory towards virusescontaining reverse transcriptases such as HIV-I. Our results alsodemonstrate the discovery that the most inhibitory oligonucleotide is ahetero sequence that has a DNA sequence corresponding to the 3'-terminalsequence of the human lysine tRNA located at the primer binding site ofthe HIV genome.

The compounds according to the present invention may be administeredtransdermally to mammalian host species having pathological conditionsbrought about by viruses, and other causative agents having a reversetranscriptase requirement for their transportation into the mammaliancell (infection), replication, or genetic expression. In theseinstances, the compounds may be formulated in suitable compositionsdetermined by the intended means of administration, according to methodsand procedures well-known to those skilled in the art. These compoundsaccording to the present invention may be further modified to enhancetransport into cells or to target specific tissues or organs by linkageof the compounds with steroids, sugars, peptides, nucleotides, lipids,or their derivatives. As used herein, the term "transdermal" is to beconsidered in its broadest meaning, that is administration across anepithelial layer of cells. As such, the term is appropriately used todesignate topical, oral, pernasal, intravenous, intramuscular, and othermethods of administration. For example, the compounds suitable for usein this invention may be formulated either individually or with other"active agents" or compounded with various conventional bases intopreparations such as creams, ointments, gels, lotions, tablets, orpharmaceutical solutions for injection or sprays depending upon thedesired mode of administration to the individual. In manufacturing thesepreparations, the composition may also be mixed with conventionalthickening agents, emollients, surfactants, pigments, perfumes,preservatives, fillers, and emulsifiers, all of which are well known andconventionally used in the formulation of transdermal preparations.Typically, these nonactive ingredients; will make up the greater part ofthe final preparation. Preferably, the compositions would bemanufactured to allow for slow-release or timed-release delivery. Dosageto be given would, of course, depend upon the route of administration,the administration vehicle, and the degree and severity of the conditionto be treated. In each instance, a minimal amount sufficient forbringing about the inhibition of the desired reverse transcriptaseenzyme would be administered.

Thus while we have illustrated and described the preferred embodiment ofour invention, it is to be understood that this invention is capable ofvariation and modification and we therefore do not wish to be limited tothe precise terms set forth, but desire to avail ourselves of suchchanges and alterations which may be made for adapting the invention tovarious usages and conditions. Accordingly, such changes and alterationsare properly intended to be within the full range of equivalents, andtherefore within the purview of the following claims.

Having thus described our invention and the manner and process of makingand using it in such full, clear, concise, and exact terms so as toenable any person skilled in the art to which it pertains, or with whichit is most nearly connected, to make and use the same;

We claim:
 1. An oligonucleotide of at least 2 nucleotides in length andhaving a 5'-terminal nucleotide of the formula: ##STR9## and a3'-terminal nucleotide of the formula: ##STR10## and wherein the 5'- and3'-terminal nucleotides of oligonucleotides containing at least threenucleotides are separated by a series of nucleotides of the formula:##STR11## wherein each individual R is hydrogen or a blocking group;wherein each individual A is hydrogen, hydroxyl, halogen, SH, NH₂, azideor OR₄ wherein R₄ is a blocking group; wherein each individual B is apurine or pyrimidine nucleotide base; and wherein each nucleotide in theoligonucleotide is connected to the preceding and following nucleotideby an internucleotide linkage; and wherein the oligonucleotide has atleast one phosphorodithioate internucleotide linkage having thestructure: ##STR12##
 2. An oligonucleotide according to claims 1 whereinA is hydrogen.
 3. An oligonucleotide according to claim 1 wherein A ishydroxyl.
 4. An oligonucleotide according to claim 1 wherein B isselected from the group consisting of cytosine, adenine, guanine, andthymine.
 5. An oligonucleotide according to claim 4 which is ahomopolymer.
 6. An oligonucleotide according to claim 5 in which each Bis cytosine.
 7. An oligonucleotide according to claim 6 which isd(CxCxCxCxCxCxCxCxCxCxCxCxCxC) wherein C is cytosine and x is aphosphorodithdate internucleotide linkage.
 8. An oligonucleotideaccording to claim 6 which is d(CpCxCpCpCpCpCpCpCpCpCpCpCxCpC) wherein Cis cytosine, p is a phosphate internucleotide linkage, and x is aphosphorodithioate internucleotide linkage.
 9. An oligonucleotideaccording to claim 6 which is d(CxCpCxCpCxCpCxCpCxCpCxCpCxCpC) wherein Cis cytosine, p is a phosphate internucleotide linkage, and x is aphosphorodithioate internucleotide linkage.
 10. An oligonucleotideaccording to claim 6 which is d(TxTxTxTxTxTxTxTxTxTxTxTxTxT) wherein Tis thymine, and x is a phosphorodithioate internucleotide linkage. 11.An oligonucleotide according to claim 5 which isd(AxAxAxAxAxAxAxAxAxAxAxAxAxA) wherein A is adenine, and x is aphosphorodithioate internucleotide linkage.
 12. An oligonucleotideaccording to claim 6 which is a heteropolymer.
 13. An oligonucleotideaccording to claim 12 which is d(GxAxTxTxCxAxGxCxTxAxGxTxCxCxA) whereinG is guanine, A is adenine, T is thymine, C is cytosine, and x is aphosphorodithioate internucleotide linkage.
 14. An oligonucleotideaccording to claim 12 which is d(GxCxTxAxCxGxGxCxTxCxGxCxTxG) wherein Gis guanine, A is adenine, T is thymine, C is cytosine, and x is aphosphorodithioate internucleotide linkage.
 15. An oligonucleotideaccording to claim 12 which is d(CxTxGxTxTxCxGxGxGxCxGxCxCxA) wherein Gis guanine, A is adenine, T is thymine, C is cytosine, and x is aphosphorodithioate internucleotide linkage.
 16. An oligonucleotideaccording to claim 1 wherein the oligonucleotide contains from 2 to 62nucleotides.
 17. An oligonucleotide according to claim 1 wherein R ishydrogen.
 18. An oligonucleotide according to claim 1 wherein R is ablocking group.
 19. An oligonucleotide according to claim 1 which is ofat least 2 nucleotides in length, and wherein each B is independentlyvaried and wherein one B is a purine and the second B is a pyrimidine.